Antiangiogenic effects of the quinoline-3-carboxamide linomide

Linomide (N-phenylmethyl-1,2-dihydro-4-hydroxyl-1-methyl-2-oxoquinoline-3-carboxa mide) has a reproducible in vivo antitumor effect against a series of both androgen responsive and independent Dunning R-3327 rat prostatic cancers. This antitumor effect of linomide is host mediated. One possible mechanism involving the host is that linomide has antiangiogenic activity. An indication that linomide treatment has antiangiogenic activity is the observation that prostatic cancers from linomide treated rats have more focal necrosis than sized matched tumors from untreated rats. To directly test if linomide has antiangiogenic activity, a newly developed Matrigel based quantitative in vivo angiogenic assay was used. These experiments demonstrated that linomide has dose dependent, antiangiogenic activity in vivo in the rat. Additional studies demonstrated that due to its antiangiogenic activity, linomide treatment of rats bearing prostate cancers resulted in a more than 40% decrease in tumor blood flow. Blood flow to a variety of non-tumor bearing organs was not decreased suggesting that linomide selectively inhibits angiogenesis and does not induce loss of established blood vessels. Using as a model the response of human umbilical vein endothelial cells to linomide treatment in a variety of in vitro assays, linomide was demonstrated to have cytostatic but not cytotoxic effect on human umbilical vein endothelial cells at a medium concentration of > or = 100 micrograms/ml. In addition, both endothelial cell chemotactic migration and invasion are steps in angiogenesis inhibited by linomide treatment.     

Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate

Vascular endothelial growth factor (VEGF) is a potent and selective vascular endothelial cell mitogen and angiogenic factor. VEGF expression is elevated in a wide variety of solid tumors and is thought to support their growth by enhancing tumor neovascularization. To block VEGF-dependent angiogenesis, tumor cells were transfected with cDNA encoding the native soluble FLT-1 (sFLT-1) truncated VEGF receptor which can function both by sequestering VEGF and, in a dominant negative fashion, by forming inactive heterodimers with membrane-spanning VEGF receptors. Transient transfection of HT-1080 human fibrosarcoma cells with a gene encoding sFLT-1 significantly inhibited their implantation and growth in the lungs of nude mice following i.v. injection and their growth as nodules from cells injected s.c. High sFLT-1 expressing stably transfected HT-1080 clones grew even slower as s.c. tumors. Finally, survival was significantly prolonged in mice injected intracranially with human glioblastoma cells stably transfected with the sflt-1 gene. The ability of sFLT-1 protein to inhibit tumor growth is presumably attributable to its paracrine inhibition of tumor angiogenesis in vivo, since it did not affect tumor cell mitogenesis in vitro. These results not only support VEGF receptors as antiangiogenic targets but also demonstrate that sflt-1 gene therapy might be a feasible approach for inhibiting tumor angiogenesis and growth.     

Bulge ductility of several occlusal contact measuring paper-based and plastic-based sheets

During angiogenesis new blood vessels sprout from an existing vascular bed. This is a prerequisite for tumor growth beyond a certain size and for metastasis formation. Tumors produce a number of cytokines. The development of in vivo bio-assays for angiogenesis and in vitro analysis of endothelial cells permits characterization of these different cytokines concerning their role in angiogenesis. Some cytokines act mitogenically on endothelial cells, others have chemotactic activity or induce tube formation and some have multiple functions. A few factors are chemotactic for macrophages, infiltrating the tumor and secreting further angiogenic cytokines. Another important role in the process of angiogenesis is played by the extracellular matrix. Proteases secreted by all cell types involved (tumor cell, endothelial cell, macrophages) degrade the extracellular matrix, thereby releasing and activating angiogenic factors sequestered in the extracellular matrix. Thus tumor cells, macrophages and extracellular matrix release cytokines which together act on endothelial cells, resulting in the growth and infiltration of new blood vessels into the tumor.     

Tumoral angiogenesis: physiopathology, prognostic value and therapeutic perspectives

INTRODUCTION: Angiogenesis activation plays a crucial role in tumoral growth and metastases dissemination. This review summarizes and analyzes current knowledge on molecular mechanisms related to angiogenesis and the prognostic value of its effectors. It also focuses on the therapeutical relevance of various drugs that might inhibit angiogenesic processes. CURRENT KNOWLEDGE AND KEY POINTS: Tumor angiogenesis involves complex interactions between tumoral, stromal, endothelial cells, fibroblasts and the extracellular matrix. Normal and malignant angiogenesis depends on the balance of proangiogenic and antiangiogenic factors. Endothelial cells are activated by growth factors, such as Vascular Endothelial Growth Factor (VEGF), and proliferate; they release proteases able to induce degradation of the basement membrane and extracellular matrix, and undergo migration and tubulogenesis. Angiostatin and endostatin are two powerful inhibitors of angiogenesis in experimental models. Assessment of intratumoral microvessel density and quantification of angiogenic factors, including VEGF, are of prognostic value in most cancers, particularly in breast cancer. However, the use of these prognosis markers in clinical practice is still controversial due to the lack of prospective studies and to technical limits inherent to the scoring and standardization of immunohistochemical methods. FUTURE PROSPECTS AND PROJECTS: Better understanding of the molecular basis of angiogenesis allows the development of new therapeutical strategies. Biochemical targets of antiangiogenic therapy are: the interaction between angiogenic factors and their receptors; the interaction of endothelial cells with the extracellular matrix; and intracellular signaling pathways. Angiogenesis inhibitors may not cause tumor regression, but inhibit cellular growth and produce "disease dormancy". Extensive phase I to III clinical trials involving antiangiogenesis therapy are in progress.     

Vascular endothelial growth factor expression in head and neck squamous cell carcinoma

BACKGROUND: Angiogenesis is an essential process required for growth and metastasis in cancer. In breast, gastric, and prostate cancer, vascular endothelial growth factor (VEGF) has been implicated in angiogenesis; however, little is known about VEGF in HNSCC. In this study, we hypothesize that VEGF is present in elevated levels in HNSCC and may therefore play a role in promoting angiogenesis. METHODS: We obtained tumor tissue from 63 HNSCC patients undergoing primary resection. All tissue samples were analyzed by immunohistochemistry (IHC) techniques for the presence and localization of VEGF; however, only 36 had sufficient amounts of tissue for quantitative analysis of VEGF by ELISA. Nine control specimens taken from patients undergoing uvulopalatopharyngoplasty were also analyzed. RESULTS: In all 63 of our patient samples we found VEGF to be present and localized to the cancer cells and endothelial cells. The poorly differentiated cancer cells stained more intensely in comparison with the well-differentiated ones. There was a 20-fold increase in the patient levels when compared with controls levels (P > or =0.05). Analysis by enzyme-linked immunosorbent assay revealed elevated mean levels of VEGF (241 +/- 326 pg/mg total protein [TP]) with a range of 2 to 1484 pg/mg TP. The control specimens had mean levels of 13 +/- 11 pg/mg TP and a range of 1 to 78 pg/mg TP. Patients who exhibited higher levels of VEGF tended to have a higher rate of disease recurrence (P < or =0.048) and shorter disease-free interval (P < or =0.05). CONCLUSIONS: The expression of VEGF in elevated levels in the HNSCC tumor microenvironment appears to be associated with more aggressive disease. Based on our results, VEGF may be an important angiogenic factor associated with cancer cells and endothelial cells in HNSCC. Further studies are needed to better define the role of VEGF in HNSCC and its role as a potential target for therapeutic intervention.     

beta Interferon inhibits HIV-1 Tat-induced angiogenesis: synergism with 13-cis retinoic acid

Kaposi's sarcoma (KS) is a highly angiogenic lesion which frequently presents as an aggressive form in HIV-infected male patients. We have previously shown that the HIV-1 Tat protein induces endothelial cell migration and invasion in vitro and a rapid angiogenic response in vivo, suggesting that it acts as a cofactor in epidemic KS. In this study we tested beta interferon (IFN beta) and retinoic acid (RA) for the inhibition of Tat-induced angiogenesis using in vivo and in vitro models. IFN beta, at a concentration above 2500 U/ml, was an effective inhibitor of Tat-stimulated growth, migration and morphogenesis of an endothelial cell line in vitro and of angiogenesis in vivo. A strong reduction of properties associated with neovascularisation was induced by 10,000 U/ml. In vivo, RA alone was on ineffective inhibitor of angiogenesis, and in vitro gave only a limited inhibition of endothelial cell growth. However, 13-cis RA used in combination with IFN beta impressively potentiated its effects. A combination of lower doses of IFN beta (2500 U/ml) and 13-cis RA induced a virtually complete inhibition of the Tat-related angiogenic phenotype both in vivo and in vitro. The potentiation of the anti-angiogenic activity of IFN beta by 13-cis RA suggests that this combination could be a useful approach for the therapy of epidemic KS.     

Specific-heat critical behavior of the structural random-field system Dy(AsxV1-x)O4

Vitamin D3 derivative 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) exerts various biological effects in cells that possess vitamin D3 receptor (VDR), including enhancement of cell differentiation and inhibition of cell proliferation. These activities of 1,25(OH)2D3 might be responsible for its anti-neoplastic effects, as shown in various experimental systems. The aim of this study was to compare the anti-angiogenic activity of 1,25(OH)2D3, retinoids, and interleukin-12 (IL-12) in an experimental tumor cell-induced angiogenesis assay in mice. Tumor cell-induced angiogenesis assay was performed in x-ray immunosuppressed BALB/c mice by intradermal injections of human tumor cell lines of different origin. The injections resulted within 3 d in a local formation of new blood vessels, and the intensity of angiogenesis correlated with the number of injected cells. Systemic treatment of the mouse recipients with 1,25(OH)2D3 significantly decreased angiogenesis, comparable to the effect of retinoids (all-trans retinoic acid [RA], 9-cis RA and 13-cis RA) and of IL-12. In vitro preincubation of the cells with all compounds (except IL-12) led to the inhibition of their angiogenic capability in vivo. Moreover, combination of 1,25(OH)2D3 and retinoids resulted in a synergistic inhibition of angiogenesis. The results strongly suggest that anti-angiogenic compounds with relatively low toxicity (e.g., 1,25(OH)2D3, retinoids, and IL-12) and their combinations could be beneficial in the treatment of some angiogenesis-associated malignancies.     

Vascular endothelial growth factor (VEGF)-C synergizes with basic fibroblast growth factor and VEGF in the induction of angiogenesis in vitro and alters endothelial cell extracellular proteolytic activity

Vascular endothelial growth factor-C (VEGF-C) is a recently characterized member of the VEGF family of angiogenic polypeptides. We demonstrate here that VEGF-C is angiogenic in vitro when added to bovine aortic or lymphatic endothelial (BAE and BLE) cells but has little or no effect on bovine microvascular endothelial (BME) cells. As reported previously for VEGF, VEGF-C and basic fibroblast growth factor (bFGF) induced a synergistic in vitro angiogenic response in all three cells lines. Unexpectedly, VEGF and VEGF-C also synergized in the in vitro angiogenic response when assessed on BAE cells. Characterization of VEGF receptor (VEGFR) expression revealed that BME, BAE, and BLE cell lines express VEGFR-1 and -2, whereas of the three cell lines assessed, only BAE cells express VEGFR-3. We also demonstrate that VEGF-C increases plasminogen activator (PA) activity in the three bovine endothelial cell lines and that this is accompanied by a concomitant increase in PA inhibitor-1. Addition of alpha2-antiplasmin to BME cells co-treated with bFGF and VEGF-C partially inhibited collagen gel invasion. These results demonstrate, first, that by acting in concert with bFGF or VEGF, VEGF-C has a potent synergistic effect on the induction of angiogenesis in vitro and, second, that like VEGF and bFGF, VEGF-C is capable of altering endothelial cell extracellular proteolytic activity. These observations also highlight the notion of context, i.e., that the activity of an angiogenesis-regulating cytokine depends on the presence and concentration of other cytokines in the pericellular environment of the responding endothelial cell.     

Regulation of local host-mediated anti-tumor mechanisms by cytokines: direct and indirect effects on leukocyte recruitment and angiogenesis

The regulation of tumor growth by cytokine-induced alterations in host effector cell recruitment and activation is intimately associated with leukocyte adhesion and angiogenic modulation. In the present study, we have developed a novel tumor model to investigate this complex series of events in response to cytokine administration. Gelatin sponges containing recombinant human basic fibroblast growth factor (rhFGFb) and B16F10 melanoma cells were implanted onto the serosal surface of the left lateral hepatic lobe in syngeneic C57BL/6 mice. The tumor model was characterized by progressive tumor growth initially localized within the sponge and the subsequent development of peritoneal carcinomatosis. Microscopic examination of the sponge matrix revealed well developed tumor-associated vascular structures and areas of endothelial cell activation as evidenced by leukocyte margination. Treatment of mice 3 days after sponge implantation with a therapeutic regimen consisting of pulse recombinant human interleukin-2 (rhIL-2) combined with recombinant murine interleukin-12 (rmIL-12) resulted in a marked hepatic mononuclear infiltrate and inhibition of tumor growth. In contrast to the control group, sponges from mice treated with rhIL-2/rmIL-12 demonstrated an overall lack of cellularity and vascular structure. The regimen of rhIL-2 in combination with rmIL-12 was equally effective against gelatin sponge implants of rhFGFb/B16F10 melanoma in SCID mice treated with anti-asialo-GM1 in the absence of a mononuclear infiltration, suggesting that T, B, and/or NK cells were not the principal mediators of the anti-tumor response in this tumor model. The absence of vascularity within the sponge after treatment suggests that a potential mechanism of rhIL-2/rmIL-12 anti-tumor activity is the inhibition of neovascular growth associated with the establishment of tumor lesions. This potential mechanism could be dissociated from the known activities of these two cytokines to induce the recruitment and activation of host effector cells. Moreover, this model provides a unique opportunity to study the cellular and molecular mechanism(s) underlying both tumor angiogenesis and leukocyte recruitment to metastatic lesions.     

Three distinct D-amino acid substitutions confer potent antiangiogenic activity on an inactive peptide derived from a thrombospondin-1 type 1 repeat

Mal II, a 19-residue peptide derived from the second type 1 properdin-like repeat of the antiangiogenic protein thrombospondin-1 (TSP-1), was inactive in angiogenesis assays. Yet the substitution of any one of three L-amino acids by their D-enantiomers conferred on this peptide a potent antiangiogenic activity approaching that of the intact 450-kDa TSP-1. Substituted peptides inhibited the migration of capillary endothelial cells with an ED50 of 8.5 nM for the D-Ile-15 substitution, 10 nM for the D-Ser-4 substitution, and 0.75 nM for the D-Ser-5 substitution. A peptide with D-Ile at position 15 could be shortened to its last seven amino acids with little loss in activity. Like whole TSP-1, the Mal II D-Ile derivative inhibited a broad range of angiogenic inducers, was selective for endothelial cells, and required CD36 receptor binding for activity. A variety of end modifications further improved peptide potency. An ethylamide-capped heptapeptide was also active systemically in that when injected i.p. it rendered mice unable to mount a corneal angiogenic response, suggesting the potential usefulness of such peptides as antiangiogenic therapeutics.     

Breast cancer increases initiation of angiogenesis without accelerating neovessel growth rate

BACKGROUND: Recurrence and mortality rates in patients with breast cancer correlate with the degree of tumor angiogenesis (angiogenic index). We have developed a novel angiogenesis model by using disks of fresh human placental vein that initiate an angiogenic response and exhibit linear radial capillary growth in culture. We hypothesized that the addition of human breast cancer cells to this human placental vein angiogenesis model would increase the incidence of angiogenesis and accelerate the rate of neovessel growth compared with vein disk cultured without tumor cells. METHODS: To test this hypothesis, vein explants from seven human placentas were incorporated into clots of 0.3% fibrin in Medium 199 and fetal bovine serum with or without 1.5 x 10(5) T-47D (n = 6 placentas) or MCF-7 (n = 1 placenta) breast cancer cells. Statistical differences between the experimental (with breast cancer cells) and control (no added cells) cultures were determined by repeated measures ANOVA. RESULTS: The proportion of disks exhibiting neovessel growth (initiation) by day 12 was significantly increased in the presence of T-47D cells (p < 0.05 at day 12, p < 0.001 at day 15). No statistical difference was seen in rates of neovessel growth (millimeters per day). Similar results were seen with MCF-7 cells. CONCLUSIONS: Tumor enhancement of angiogenesis may occur by increased initiation of the angiogenic response. Subsequent vessel growth rates may be tumor independent. We predict that effective antiangiogenic therapies will block a tumor's ability to augment angiogenesis initiation rather than subsequent neovessel growth.     

Effects of tamoxifen and interferon-beta or the combination on tumor-induced angiogenesis

Inhibition of angiogenesis by anti-tumor agents may play a role in tumor growth arrest. Tamoxifen and interferon-alpha/beta (IFN-alpha/beta) exhibit potentiated anti-proliferative activity against tumor cells. However, additional host-mediated effects such as modulation of angiogenesis may also inhibit tumor growth in vivo. The effect of tamoxifen and IFN-beta on angiogenesis induced by 2 human tumors, MCF-7 breast carcinoma (estradiol dependent) and NIH-OVCAR-3 ovarian carcinoma (estradiol independent), was assessed. Treatment of nude mice bearing MCF-7 tumors with tamoxifen resulted in a 68% decrease in the number of vessels at the tumor periphery. Treatment with IFN-beta yielded a 33% reduction. Treatment of nude mice bearing NIH-OVCAR-3 tumors with tamoxifen resulted in a 73% decrease in the number of vessels. Treatment with IFN-beta yielded a 57% reduction. Combination treatment resulted in augmented anti-angiogenic effects. As single agents, both tamoxifen and IFN-beta inhibited xenograft tumor growth. Ten weeks of tamoxifen treatment resulted in growth inhibition of MCF-7 and NIH-OVCAR-3 carcinomas by 85% and 66%, respectively. Ten weeks of IFN-beta treatment resulted in inhibition of growth of MCF-7 and NIH-OVCAR-3 carcinomas by 67% and 88%, respectively. The combination of tamoxifen and IFN-beta completely prevented growth of MCF-7 and NIH-OVCAR-3 carcinomas. The anti-angiogenic effects of tamoxifen and IFN-beta were additive. Inhibition of angiogenesis was detectable before measurable effects on tumor volume in both MCF-7 and NIH-OVCAR-3 tumors. Potentiation of anti-angiogenic effects by tamoxifen and IFN-beta, possibly resulting from enhanced IFN-induced gene expression, may contribute to anti-tumor activity in both estradiol-dependent and estradiol-independent tumors in vivo.     

Angiogenic activity of osteopontin-derived peptide SVVYGLR

Angiogenesis plays an important role in various pathological conditions as well as some physiological processes. Although a number of soluble angiogenic factors have been reported, extracellular matrix also has crucial effect on angiogenesis through interaction with endothelial cells. Since recent reports showed osteopontin had some angiogenic activity, the effect of the SVVYGLR peptide, novel binding motif in osteopontin molecule, on angiogenesis was examined in this study. Synthetic peptide SVVYGLR did not have proliferative effect on endothelial cells but adhesion and migration activity to endothelial cells. Furthermore, SVVYGLR had as potent activity for tube formation in three-dimensional collagen gel as vascular endothelial growth factor which is known to be the strongest angiogenic factor. Electron microscopical analysis showed a number of microvilli on the endothelial luminar surface and tight junction formation in the luminar intercellular border between endothelial cells, indicating SVVYGLR induced cell porarity and differentiation of endothelial cells. This small peptide might be expected to stimulate angiogenesis to improve some ischemic conditions in the future because of some advantages due to smaller molecular weight.     

Primary tumor size-dependent inhibition of angiogenesis at a secondary site: an intravital microscopic study in mice

Some primary tumors are capable of suppressing the growth of their metastases by presumably generating antiangiogenic factors such as angiostatin. We hypothesized that the amount of inhibitor(s) released by a tumor increases with tumor growth. We tested this hypothesis by evaluating the relationship between the size of a primary tumor and its ability to inhibit angiogenesis at a secondary site. Furthermore, we characterized the effects of the primary tumor on physiological properties of newly formed vessels at the secondary site. Angiogenesis and physiological properties were measured using intravital microscopy of angiogenic vessels in the gels containing basic fibroblast growth factor placed into cranial windows of immunodeficient mice bearing human prostatic carcinoma (PC-3) in their flank. The PC-3 tumor inhibited angiogenesis in the gels, and surgical resection of tumor reversed this inhibition. The inhibition of angiogenesis 20 days after gel implantation (range, 0-83%) correlated positively (r = 0.625; P < 0.008) with the tumor size on the day of gel implantation (range, 19-980 mm3). The primary tumor also suppressed leukocyte-adhesion in angiogenic vessels, thus helping them evade the immune recognition. These results provide an additional rationale for combining antiangiogenic treatment with local therapies.     

Inhibition of tumor growth and angiogenesis by vitamin D3 agents in murine renal cell carcinoma

PURPOSE: To investigate the effect of active vitamin D3(VD) agents on tumor growth and metastasis. MATERIALS AND METHODS: BALB/c mice were inoculated with murine renal cancer Renca and graded doses of 1,25-dehydrovitamin D3 or 1- hydrovitamin D3 were given intraperitoneally every other day beginning on day 1, 3, or 7 and ending on day 9, 11, or 15. Direct cytocidal activity and angiogenic activity were evaluated by 48-hour MTT assay and by the colorimetric method, respectively. RESULTS: Both VD agents inhibited tumor growth and prolonged the life span of Renca-bearing mice in a dose-dependent manner and both suppressed tumor growth in athymic mice and euthymic mice with eliminated NK activity. Marginal body-weight loss without appreciable hypercalcemia was observed in mice given VD agents. When treatment was delayed on day 7, the VD agents failed to inhibit tumor growth. The MTT assay showed no direct cytotoxicity of VD agents on Renca. Tumor angiogenesis was inhibited to 46 to 30% of the control level by VD agents. Furthermore, VD agents reduced pulmonary and hepatic foci in the metastatic models. CONCLUSIONS: These results suggest that VD agents may be effective as a treatment for renal cell carcinoma, especially when micrometastases are involved.     

The prevalence of rheumatic diseases in a Filipino urban population: a WHO-ILAR COPCORD Study. World Health Organization. International League of Associations for Rheumatology. Community Oriented Programme for the Control of the Rheumatic Diseases

Recent analysis of bladder tumors has correlated expression of the neurokine midkine (MK) with poor patient survival. To examine a role for MK and the related pleiotrophin (PTN) in tumorigenesis, they were overexpressed in MCF-7 breast carcinoma cells. Expression had no effect on in vitro growth but conferred a growth advantage in vivo. Enhanced tumor growth correlated with increased vascular density and endothelial proliferation, implicating an angiogenic role for MK and PTN. Angiogenic activity of MK and PTN was confirmed in the rabbit corneal assay. Our data therefore identify two novel targets for antiangiogenic drug development.     

Omega-3 polyunsaturated fatty acid levels in the diet and in red blood cell membranes of depressed patients

The objective of this study was to clarify the possible angiogenesis-promoting factors from human trophoblasts in early stage gestation. The existence of angiogenic growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the condition medium from human villous trophoblasts was determined. Biological activity of angiogenic growth factors released by trophoblasts was examined using vascular endothelial cell lines. The condition medium from trophoblasts enhanced the growth of endothelial cells. Although cultured trophoblasts exhibited immunoreactive products for both bFGF and VEGF in the cytoplasm, only bFGF was detected in the condition medium by ELISA. The growth-enhancing activity of the condition medium was eliminated completely by the addition of anti-bFGF antibody but not with anti-VEGF antibody. Thus, trophoblastic cells seem to play an important role in extensive angiogenesis occurring in early gestation, mainly by releasing bFGF but not VEGF.     

Therapeutic usefulness of wild-type p53 gene introduction in a p53-null anaplastic thyroid carcinoma cell line

Anaplastic thyroid carcinomas very often harbor the mutations in the tumor suppressor gene p53. We have previously shown that wild-type (wt) p53 gene introduction led to cell growth arrest, but not apoptosis, in p53-null anaplastic thyroid carcinoma cells. The present studies were designed to evaluate other therapeutic effects of wt-p53 gene introduction on p53-null thyroid carcinoma cells, as chemo- and radiosensitization and inhibition of angiogenesis have also been described recently as additional therapeutic advantages of wt-p53 gene introduction in tumor cells with p53 mutations. A p53-null anaplastic thyroid carcinoma cell line, FRO, and a FRO subline stably expressing a temperature-sensitive (ts) mutant of p53 (p53Val138), tsFRO, were used. ts-p53 functions as mutant and wt at nonpermissive (37 C) and permissive (32 C) temperatures, respectively. tsFRO showed a prolonged cell doubling time compared to parental FRO when cultured at 32 C, but the cell growth rate was similar between FRO and tsFRO at 37 C. The cytotoxic and clonogenic assays demonstrated that although the sensitivity to three different anticancer agents (cisplatin, 5-fluorocytosine, and doxorubicin) was unaltered, radiosensitivity was enhanced in tsFRO compared to FRO at 32 C. Unexpectedly, in studies on angiogenesis, expression levels of vascular endothelial growth factor (an angiogenic factor) messenger ribonucleic acid were similar between FRO and tsFRO, and thrombospondin-1 (an antiangiogenic factor) messenger ribonucleic acid and protein levels were about 2.5-fold lower in tsFRO than FRO at 32 C, although any difference could not be detected in their ability to inhibit in vitro angiogenesis with the culture medium conditioned by tsFRO and FRO at 32 C. These results suggest that p53-defective thyroid carcinomas may benefit from the combination of p53 gene therapy and radiotherapy. However, further study will be necessary to clarify the pathological significance of thrombospondin-1 in angiogenesis and thyroid tumor growth.     

Significance of vessel count and vascular endothelial growth factor and its receptor (KDR) in intestinal-type gastric cancer

Angiogenesis is essential for tumor growth and metastasis and depends on the production of angiogenic factors by host and/or tumor cells. The role of angiogenesis and angiogenic factor expression in intestinal- and diffuse-type gastric cancer are undefined. Archival specimens of 51 intestinal-type and 38 diffuse-type human gastric carcinomas were examined for tumor vessel counts, angiogenic factor expression, and the presence or absence of angiogenic factor receptors on tumor endothelium using antibodies against vascular endothelial growth factor (VEGF) and its receptors (KDR and flt-1), basic fibroblast growth factor (bFGF) and its receptors (bek and flg), and factor VIII (endothelial cells). Vessel count and VEGF and bFGF expression were higher in intestinal-type than in diffuse-type gastric cancers (P = 0.01, P < 0.001, and P < 0.001, respectively). Similarly, vessel count and VEGF expression were higher in patients with liver metastasis than in patients with peritoneal dissemination (P = 0.003 and P = 0.01, respectively). Vessel count correlated with VEGF expression and the presence of endothelial KDR in intestinal-type gastric cancer (P = 0.003 and P = 0.02, respectively) but not diffuse-type gastric cancer. Vessel count, VEGF expression, and presence of endothelial KDR increased with increasing stage of disease in intestinal-type gastric cancer but not diffuse-type gastric cancer. The expression of bFGF and its receptors did not correlate with vessel count in either cancer type. These findings suggest that the pattern of metastasis in intestinal-type gastric cancer is angiogenesis dependent. The correlation of VEGF expression and its endothelial receptor with vessel count and stage of disease suggests that VEGF is at least one of the factors responsible for the induction of angiogenesis in intestinal-type gastric cancer.